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Journal of Chromatography A, 1216 (2009) 2567–2573
Contents lists available at ScienceDirect
Journal of Chromatography A
journa l homepage: www.e lsev ier .com/ locate /chroma
nzyme enhanced quantitative determination of multiple DNA targets based onapillary electrophoresis
uemei Lia,b, Zhiming Zhanb, Shusheng Zhangb,∗, Hongyuan Chena,∗∗
Department of Chemistry, Nanjing University, Nanjing 210093, ChinaKey Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology,ingdao 266042, China
r t i c l e i n f o
rticle history:eceived 15 October 2008ccepted 5 January 2009vailable online 15 January 2009
a b s t r a c t
In the current paper, enzyme enhanced simultaneous quantitative determination of multiple DNA targetsbased on capillary electrophoresis (CE) was described. We used three biotin-modified DNA probes, whichreacted with avidin-conjugated horseradish peroxidase (avidin-HRP) conjugate to obtain the HRP labeled
eywords:apillary electrophoresisNAultianalyte determination
probes, to hybridize with three corresponding targets. The resulting mixture containing double-strandDNA (dsDNA)-HRP, excess single-strand DNA (ssDNA)-HRP and remaining avidin-HRP was separated bycapillary electrophoresis, and then the system of HRP catalyzing H2O2/o-aminophenol (OAP) reaction wasadopted. The catalytic product was detected with electrochemical detection. With this protocol, the limitsof quantification for the hybridization assay of 21-, 39- and 80-mer DNA fragments were of 1.2 × 10−11,
−11 −11 M, re
nzyme enhancementlectrochemical detection2.4 × 10 and 3.0 × 10cross-reaction.
. Introduction
Sequence-specific detection of DNA targets is of central impor-ance to the diagnosis and treatment of genetic diseases, for theetection of infectious agents, and for forensic investigations [1–3].ignificant progress has been made for single analyte detection4–9]. Recent activity has focused on the development of hybridiza-ion assays that permit simultaneous determination of multipleNA targets [10–18], which is needed in more advanced applica-
ions. The ability to quantify multiple nucleic acid sequences inarallel using a single sample allows researchers and clinicians tobtain high-density information with minimal assay time, sampleolume, and cost [19].
There are two main approaches used for multiplexing: pla-ar surface arrays and suspension array. The first protocol, knowns DNA microarray, is best suited for applications requiring
ltra-high-density analysis [11,20]. Despite these successes, DNAiochip fabrication methods remain a challenge for creating cost-ffective devices. It often requires the additional capability ofulticolor labeling, which has several disadvantages. An alter-ative to biochips is an approach based on DNA probes bound
∗ Corresponding author. Tel.: +86 53284022750; fax: +86 532 84022750.∗∗ Corresponding author. Tel.: +86 025 83594862; fax: +86 025 83594862.
E-mail addresses: [email protected] (S. Zhang), [email protected]. Chen).
021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.01.019
spectively. The multiplex assay also provided good specificity without any
© 2009 Elsevier B.V. All rights reserved.
to microspheres [21,22]. In comparison with planar DNA chips,encoded-bead technology is expected to be more flexible in targetselection, faster in binding kinetics, and less expensive to produce.Although the production of such microspheres is less costly andelaborate than biochip array production, the analysis of micro-spheres in solution is more complex.
Capillary electrophoresis (CE) is a powerful separation tech-nique that has been very useful in the analysis of biological samplesas it has provided for methods with high selectivity, small sam-ple consumption, ease of use, high separation efficiencies, andshort analysis times [23,24]. These advantages meet the require-ments for the study of biomolecular interactions and CE has beenregarded as one of the useful analytical tools for biological samples,such as immunoassay [25–27], proteins [28,29], and DNA [30–32].Especially, affinity capillary electrophoresis (ACE), which is basedon hybridization, is the most powerful tool in CE and can attainselective separations of biomolecules [33,34]. Though CE has beenextensively applied to single analyte analysis, a limited amountof research has reported on the simultaneous detection of mul-tiple analytes. Wang et al. have developed a chip-CE strategy forsimultaneous glucose and insulin measurements, comprising enzy-matic and immunological assays on a single-channel microfluidicdevice with an amperometric detector [35]. Le and co-workers
demonstrated a tunable aptamer capillary electrophoresis tech-nique to the simultaneous determination of pM levels of fourproteins in a single analysis [36]. However, there is no report on thesimultaneous determination of multiple DNA targets by capillaryelectrophoresis.
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In this paper, enzyme enhanced quantitative determinationf multiple DNA targets based on CE-electrochemical detec-ion (CE-ED) was described. We demonstrated the simultaneousetection of 21-, 39- and 80-mer DNA targets in one singleample. Three corresponding biotin-modified DNA probes, whicheacted with avidin-conjugated horseradish peroxidase (avidin-RP) conjugate to obtain the HRP labeled probes, hybridizedith these three targets. The resulting mixture containing hybridsDNA-HRP, excess ssDNA-HRP and remaining avidin-HRP waseparated by CE. The enzymatic product, 2-aminophenoxazine-3-ne (AP), produced from the oxidation of o-aminophenol (OAP)ith H2O2 catalyzed by HRP, was amperometrically detected at a
t electrode at the outlet of the reaction capillary. We extendedingle analyte detection of DNA based on CE to multiplex DNAssay.
. Experimental
.1. Chemicals and reagents
Avidin-HRP was obtained from cell chip biotech Co., Ltd.Beijing, China). All oligonucleotides in the present study wereurchased from SBS Genetech Co., Ltd. (Beijing, China), andequences were listed in Table 1. Capture DNA probes were biotin-unctionalized at the 5’ end, and reacted with avidin-HRP toield ssDNA-HRP adducts. The running buffer consisted of 1.0 mM2O2, 1.0 × 10−2 M Britton–Robinson buffer (BR buffer, the mix-
ure of 0.98 g H3PO4 + 0.60 g HAc + 0.62 g H3BO3 was diluted toL, and adjusted to pH 6.0 with 0.2 M NaOH), and 4 mM MgCl2.1.0 × 10−2 M stock solution of OAP was prepared by dissolving
n appropriate amount of OAP in water. All other reagents weref analytical grade and were used as received. All solutions wererepared with double-distilled water and filtered through 0.45 �mellulose acetate membrane filters (Shanghai Yadong Resin Co., Ltd.,hanghai, China) before use.
.2. Equipment and conditions
The CE-ED system used in this work was purchased fromi’an REMEX Analyse Instrument Co., Ltd. (Xi’an, China), equippedith an electrochemical analyzer (Model MPI-A) perform-
ng the amperometric detection at a constant potential. Theolyacrylamide-coated fused silica capillaries (50 �m ID, 375 �mD) were obtained from Sepax Technologies Co., Ltd. (USA), to
educe the electroosmotic flow (EOF) to achieve a better separa-ion and to reduce the adsorption of DNAs by the column wall.he lengths of the separation capillary and the reaction capil-ary were of 20 and 5 cm, respectively. Electrochemical detection
as carried out with a three-electrode system consisting of at working electrode, an Ag/AgCl reference electrode and a Ptuxiliary electrode. The working electrode was cleaned by ultra-onication in ethanol and double-distilled water for 5 min beforese.
able 1ist of DNA oligomers used in this study.
ame Sequence
robe 1 5′-Biotin-GAG GAG TTG GGG GAG CAC ATT-3′
robe 2 5′-Biotin-TCC CTC AGA CCC TTT TAG TCA GTG TGG AAA ATC TCT AGC-3′
robe 3 5′-Biotin-ATC CGC CTG ATT AGC GAT ACT CAG AAG GAT AAA CTG TCC AGA Aarget 1 5′-AAT GTG CTC CCC CAA CTC CTC-3′
arget 2 5′-GCT AGA GAT TTT CCA CAC TGA CTA AAA GGG TCT GAG GGA-3′
arget 3 5′-TA GTG AAG TAT GAT GTA TTG TAG TGA TGA GTT CCA AGT TCT GGA CAG
216 (2009) 2567–2573
2.3. Multianalyte hybridization
The different biotin-modified DNA probes were mixed at roomtemperature, and reacted with avidin-HRP conjugates at room tem-perature for 20 min. In order to insure that all the ssDNA probeswere functionalized with HRP, an excess avidin-HRP was applied.After the reaction, 20 mM tris(hydroxymethy) aminomethane–HClbuffer (Tris–HCl pH 7.0) containing targets 1, 2 and 3 was thenadded. The mixture was incubated for 45 min at 38 ◦C with shakingto form double-strand DNA (dsDNA).
2.4. CE procedures
Polyacrylamide-coated fused silica capillary was rinsed with BRbuffer for 4 min at 12 kV between runs, under which the capillarywas stable. The characterization of the ssDNA and hybrid was car-ried out using CE-ED system as described previously [37]. Briefly,the resulting incubation mixture containing dsDNA-HRP, excessssDNA-HRP and remaining avidin-HRP was injected into the posi-tive end of the separating capillary with the hydrodynamic pressureat 15 cm height for 10 s and then separated. The substrate OAP wasintroduced to the end of the separation capillary by hydrodynamicpressure. dsDNA-HRP, ssDNA-HRP, and avidin-HRP catalyzed thereaction of enzyme substrate. The enzymatic product, AP, producedfrom the oxidation of OAP with H2O2 catalyzed by HRP, was amper-ometrically detected on a Pt electrode at the outlet of the reactioncapillary. The concentrations for targets ssDNA were calculated bypeak areas.
3. Results and discussion
The principle of the novel multianalyte hybridization assay wasillustrated in Fig. 1. First, a solution of three biotin-modified ssDNAprobes was mixed with avidin-HRP to obtain ssDNA-HRP conju-gates. Subsequently, the conjugates were reacted with a samplecontaining the three complementary targets DNA. Next, the result-ing mixture was introduced into the capillary from the anodicend of the separation capillary and a high electric voltage wasthen applied. The dsDNA-HRP, excess ssDNA-HRP and remainingavidin-HRP migrated toward the cathode and were separated fromeach other due to their different mobility. The substrate OAP wasintroduced to the end of the separation capillary, and dsDNA-HRP,ssDNA-HRP, and avidin-HRP catalyzed the reaction of enzyme sub-strate. The enzymatic product entered into the reaction capillaryand was amperometrically detected on a Pt electrode at the outletof the reaction capillary.
3.1. Optimization of CE conditions
The pH of the buffer has an important effect on the surface char-acteristics of the polyacrylamide-coated fused-silica capillary andthe effective electric charge of the ion. The effect of pH on the peakarea was investigated in detail and shown in Fig. 2. The peak height
Description
21-mer probe39-mer probe
CT TGG AAC TCA TCA CTA CAA TAC ATC ATA CTT CAC TA-3′ 80-mer probeComplementaryto probe 1Complementaryto probe 2
TTT ATC CTT CTG AGT ATC GCT AAT CAG GCG GAT-3′ Complementaryto probe 3
X. Li et al. / J. Chromatogr. A 1216 (2009) 2567–2573 2569
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a higher concentration of magnesium ion (8 mM) for target 3 couldbe a stable dsDNA of target 3 generated, which reduced the tail-ing of the peak comparing to the corresponding unstable one. Takeaccount of the simultaneous detection of three targets DNA, 4 mMMgCl2 was chosen as a compromise.
ig. 1. Schematic principle of multiple DNA assay. (a) Biotin-modified ssDNA probeparation of the mixture containing dsDNA-HRP, excess ssDNA-HRP and remaining
f the complex increased as pH increased from 5.0 to 7.0, due to theigher yields of the hybridization at higher pH. The results showedhat pH 6.0 was the best pH value at which the peaks were sharpnd symmetrical. The possible reason is that, as a small molecularlycoprotein, the isoelectric point of HRP is 6.5. When it is belowH 6.0, the hybridization of the DNA was not completed. When theH is over 6.0, it would be too far from the optimal condition of HRPatalysis activity (pH 3–4) [38].
Magnesium ion can increase the binding energy between twoNA strands due to a reduction in the electrostatic repulsionetween them [39]. Therefore, when the magnesium ion concen-ration is high, the probe DNA may hybridize with both targetnd non-target DNA, while in low magnesium ion concentrations,he ligand DNA may not hybridize with any DNA, keeping theingle-strand structure. Consequently, in hybridization-based mul-ianalyte assay, the optimization of magnesium ion concentrations essential to differentiate target DNA from non-target DNA. Theunning buffer solution containing 4 or 8 mM MgCl2 was used tonalyse targets 1, 2 and 3. The results showed that the separationerformance was better at 4 mM MgCl2 for targets 1 and 2, while
etter at 8 mM MgCl2 for target 3. This is probably because theigher order structure of 80-mer DNA hindered the binding of someases to the probe DNA, and reduced the binding energy. Higheragnesium ion increased the binding energy and thus facilitatedig. 2. Effect of buffer pHs on the peak areas. (�) Target 1, (�) target 2, (�) target 3. Allrobe and target concentrations were of 3.0 × 10−10 and 1.1 × 10−10 M, respectively.ll analytes were prepared in the 1.0 × 10-2 M BR buffer, Ed, −0.45 V; separation volt-ge, 12 kV; separation capillary, 20 cm 50 �m ID; reaction capillary, 5 cm, 50 �m ID;njection height and time, 15 cm and 10 s.
) ssDNA-HRP conjugates, (c) hybridization with the target DNA, (d) injection andn-HRP, (e) enzyme catalytic reaction, (f) electrochemical detection.
the hybridization. Magnesium ion can also increase the stability ofdsDNA, and longer dsDNA needs higher concentration of magne-sium ion to stable it. The better separation observed in the case of
Fig. 3. Electropherograms of 3.0 × 10−10 M 21-mer probe DNA, 5.0 × 10−10 M avidin-HRP and different concentrations of target DNA. Target DNA concentrations: (a) 0, (b)1.5 × 10−10 M, (c) 1.5 × 10−10 M, (d) 3.0 × 10−10 M. All analytes were prepared in the0.2 M BR buffer at 37 ◦C. Ed, −0.45 V; separation voltage, 12 kV; separation capillary,20 cm × 50 �m ID; reaction capillary, 5 cm × 50 �m ID; injection height and time,15 cm and 10 s. Peaks: (1) free avidin-HRP, (2) ssDNA-HRP, (3) dsDNA-HRP.
2570 X. Li et al. / J. Chromatogr. A 1
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3.3. Effect of DNA lengths on electrophoretic mobility
A variety of DNA fragments with different lengths werehybridized with their complementary targets and the electro-
ig. 4. Electropherograms of different target DNA. (a) 21-mer, (b) 39-mer, (c) 80-er. The probe and target concentrations for three DNA were of 3.0 × 10−10 and
.5 × 10−10 M, respectively. Peak description was the same as in Fig. 2.
.2. Electrophoretic characterization of ssDNA and dsDNA
Direct evidence of the interactions between probe ssDNA and itsomplementary target can be produced from the electrophoreticrocedures in this study. Single-analyte assay was carried out firsto assess the sensitivity and selectivity of the new method. The lowetection limit of 1.09 × 10−11 M (S/N = 3) for HRP of the CE-ED tech-ique [37] and its good resolution for separation of ssDNA fromheir respective hybrids allowed sensitive detection of target ssDNA.dditional advantages of the CE-enzyme assay-electrochemicaletection (CE-EA-ED) technique for detection of multiple DNA tar-ets included the enzyme amplified effect, no wash steps required.
A series of electropherograms from the CE-EA-ED assay of mix-
ures containing 3.0 × 10−10 M specific probe DNA, 5.0 × 10−10 Mvidin-HRP and different concentrations of target DNA were shownn Fig. 3. The free avidin-HRP was well resolved from the ssDNA-RP conjugates. Since the electrophoretic mobility of DNA carriesig. 5. Simultaneous detection of targets 1, 2, and 3. (a) Sample containing probes 1,, and 3 interacted with avidin-HRP, (b) after hybridize with targets 1, 2, and 3. Peaksepresentation: (1) free avidin-HRP, (2) 21-mer DNA coupled avidin-HRP, (3) 21-er DNA coupled avidin-HRP hybridization with target 1, (4) 39-mer DNA coupled
vidin-HRP, (5) 39-mer DNA coupled avidin-HRP hybridization with target 2, (6) 80-er DNA coupled avidin-HRP, (7) 80-mer DNA coupled avidin-HRP hybridizationith target 3.
216 (2009) 2567–2573
in the direction opposite to that of EOF due to its negatively chargednature [40], the free avidin-HRP species have higher electrophoreticmobility compared with that of ssDNA-HRP adducts. The peaks offree avidin-HRP and ssDNA-HRP conjugates corresponded to peak1 (∼80 s) and peak 2 (∼170 s), respectively. When target DNA wasadded to form dsDNA, a new peak (peak 3) that corresponded tothe hybrid peak appeared right after peak 2 that decreased. Amongthe three HRP labeled species, the dsDNA hybrid has the highestnegative effective charge and has the highest electrophoretic mobil-ity toward the positive (injection) end. Thus the hybrid migratedslower than the ssDNA, resulting in baseline separation from ssDNA.
Fig. 6. Calibration curves of peak area versus the mixture of three targets A (�), B (�),and C (�). Experimental conditions: all probe concentrations were of 5.0 × 10−9 M.The detection procedure was carried out as described in the experimental section.
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herograms were shown in Fig. 4. The retention times of the freevidin-HRP, probes 1, 2, and 3, and their corresponding hybrids
ere of 80, 170, 190, 340, 580, 750, and 1070 s, respectively.horter DNA migrated faster than longer DNA in the separationapillary as in typical DNA separation. These observations sug-ested that the relative mobilities of these DNA hybrids wereependent upon their charge-to-mass ratios. These features facil-
ig. 7. Specificity of the probes used for the multianalyte hybridization assay. (a) Target 1arget 1,2, and 3. The concentrations for each probe DNA and target DNA were 3.0 × 10−10
216 (2009) 2567–2573 2571
itated multiplex analysis based on the differences of migrationtimes.
3.4. Simultaneous determination of three targets DNA
To demonstrate simultaneous detection of targets 1, 2, and 3,samples containing targets 1, 2, and 3 were used. As shown in Fig. 5a,
, (b) target 2, (c) target 3, (d) target 1 and 2, (e) target 1 and 3, (f) target 2 and 3, (g)and 2.0 × 10−10 M, respectively.
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hree peaks other than the free avidin-HRP peak were observed inhe electropherogram. Peaks 2, 4, and 6 corresponded to the probes, 2, and 3, respectively. After interaction with the targets, threeore peaks, peaks 3, 5, and 7, appeared as the corresponding dsDNA
ybrids (Fig. 5b). In the hybridization complexes, all the seven peaksere separated well from each other in the electropherogram and
ould be used to detect targets 1, 2, and 3 simultaneously in a singlessay.
.5. DNA quantification and detection limit
Under the conditions described above, the mixtures of threeargets can be analyzed in a quantitative fashion. The sensitiv-ty and analytical range of the multianalyte hybridization assayor all DNA fragments were performed. The relationship betweenhe hybrid peak areas and the three target DNA concentrationsas presented in Fig. 6, showing that the hybridization was in
esponse to sample mixtures containing increasing levels of threeligonucleotides. The points in the curves represent the averageeak area detected from three repeatable runs. The target 1 concen-ration from 4.0 × 10−11 to 2.0 × 10−9 M had a good linear relationith the peak area in BR buffer solution. The regression equa-
ion was y = 0.3001x + 1.2892 (x was the amount of the target DNA,0−11 M; y was the peak area, nC), and the regression coefficientf the linear curve was 0.9985. A detection limit of 1.2 × 10−11 Mf the complementary oligonucleotides can be estimated using/N = 3. The linear range (n = 7, r = 0.9996) and detection limit forarget 2 were 5.0 × 10−11 to 1.2 × 10−9 M and 2.4 × 10−11 M, withhe equation of linear regression being y = 0.2632x + 0.3417 (x washe amount of the target DNA, 10−11 M; y was the peak area,C). And the linear range (n = 7, r = 0.9995) and detection limit forarget 3 were 5.0 × 10−11 to 1.0 × 10−9 M and 3.0 × 10−11 M, withhe equation of linear regression being y = 0.2840x + 0.5451 (x washe amount of the target DNA, 10−11 M; y was the peak area,C).
Our technique was competitive with most of the other label-ree single-target [41] or fluorescent [42] and nanoparticle-basedlectrochemical multitarget DNA chip [14] as reported previ-usly. This system used only 4 �L of sample, which furthereduced the absolute number of DNA molecules required for anssay.
A series of seven parallel measurements of samples contain-ng targets 1, 2, and 3 yielded relative standard deviation of 7.2%,.4%, and 6.9% for targets 1, 2, and 3, respectively, when the con-entrations for all three DNA were 1.0 × 10−10 M. Recoveries werebtained as 89.2–108.4%, 90.7–104.3%, and 94.8–103.8% for threeargets, respectively (n = 7).
.6. Selectivity of the technique
The selectivity of this technique was based on the hybridiza-ion specificity between probe DNA and its target. The experimentsn terms of sequence specificity and absence of nonspecific bind-ng of the probes were carried out to investigate whether theres any cross-hybridization of each probe with the DNA fragmentsresent in the mixture. Each DNA fragment was hybridized with aixture of the three probes. The results of the cross-hybridization
tudies were presented in Fig. 7. There was only one target DNAequence, target 1, in the solution, and only one conjugate peakas obtained in the CE analyses (Fig. 7a). Similar selectivity wasbserved for the other DNA sequences using the corresponding tar-
et solutions (Fig. 7b and c), the individual DNA targets yieldingell-defined hybridization signals. A sample mixture containingwo or three DNA targets thus yielded the corresponding two orhree signals (Fig. 7d–g). These results showed that the probesere specific for their cognate DNA fragments and our technique
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216 (2009) 2567–2573
could simultaneously determine three targets in a single sam-ple.
4. Conclusion
A simple and rapid CE-based multiple oligonucleotide detec-tion was demonstrated for performing simultaneous three-DNAdetermination, based on three biotin-modified DNA probes. Tra-ditionally, multiple nucleic acid detection involved in the probeimmobilization on a solid phase, which was complicated and unsta-ble. While, a significant advantage of this approach was that allthe hybridizations were performed in one vial with a lower non-specific affinity, making the assay quite simple. In addition, withthe enhancement of the HRP, a higher sensitivity was achieved.The assay was not limited to the three DNA targets shown here,and the principle can be extended to the simultaneous analysisof more DNA sequences, which had different mobilities to eachother. Overall, the design and concept of multiple DNA detectionsystem presented herein provided the foundation for the develop-ment of sensitive multianalyte assay, which was useful in clinicaland research applications.
Acknowledgments
This work was supported by the Natural Science Foundation ofShandong Province (No. Y2007B31), the National Natural ScienceFoundation of China (No. 20875052), and the National High-techR&D Program (863 Program, No. 2007AA09Z113).
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